Dysregulated growth factor gene expression is associated with tubulointerstitial apoptosis and renal dysfunction.

Chronic renal disease is characterized by declining renal function, loss of intrinsic renal cells, and their replacement with fibrotic tissue. This study investigates apoptosis and its regulation in the context of chronic renal disease. RNA was extracted from renal biopsies from patients with various forms of chronic renal disease. Expression of genes of the Bcl-2 family, death receptor pathway, and growth factors were measured by reverse-transcription real-time polymerase chain reaction. Apoptosis was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling technique. Tubulointerstitial apoptosis was positively associated with tubulointerstitial injury and renal dysfunction and increased 2.3-fold per unit (U) increase in transforming growth factor beta(1) (TGFbeta(1)) mRNA (P<0.05). Conversely, a 1 U increase in epidermal growth factor (EGF) mRNA was associated with a 47% decrease in tubulointerstitial apoptosis (P<0.05). Tubulointerstitial injury was correlated with increased TGFbeta(1) and tumour necrosis factor alpha (TNFalpha) mRNA (P<0.005) and decreased EGF mRNA (P<0.05). Additionally, for a 10 U decrease in the glomerular filtration rate there was an estimated increase of 5 and 10% in TGFbeta(1) and TNFalpha mRNA, respectively (P<0.05), whereas EGF mRNA decreased by an estimated 15% (P<0.005). Therefore dysregulation of cytokine/growth factor expression plays a central role in the progression of chronic renal disease through contribution to renal cell loss, tubulointerstitial injury, and renal dysfunction.

The evaluation and treatment of pediatric oral habits.

Oral habits continue to be a common problem in pediatric dentistry. This article briefly describes the most common oral habits and presents treatment options for resulting problems.

Functional, histological, and inflammatory changes in chronically rejecting small bowel transplants.

Our aim was to develop a model of chronic rejection (CR) in small bowel allografts, and to study the changes occurring in these grafts. Small bowel transplantation was performed using the DA to AS rat strain combination. Short-term (5 mg/kg intramuscular, from days -2 to +9), or long-term cyclosporin treatment (5 mg/kg, 3 times a week until day 50) was given to prevent acute rejection. Controls were untreated allografts, DA isografts with and without cyclosporin, and normal DA and AS rats. They were followed for 50 and 100 days after transplantation. Recipients of a syngeneic graft lost weight during the first week after transplantation, but started to regain weight and kept growing thereafter. Histology showed normal bowel architecture with normal mesenteric lymph nodes and Peyers patches. Vigorous acute rejection occurred in the untreated allografts. Animals had persistent weight loss, and were killed between 6-13 days after transplantation. No clinical signs of graft-versus-host disease were seen. Histology showed end-stage acute rejection. In both cyclosporin-treated allografted groups the postoperative course was as in the isografted animals. However, all animals had histologic signs of CR by 50 and 100 days after transplantation. Changes were most prominent in the mesentery. Serositis with increased vascularity, inflammation with sclerosis, and patchy myointimal proliferation with endothelialitis of the mesenteric vessels were found. Changes in the bowel were patchy and included some thickening of the muscle coat, crypt hyperplasia, scattered necrotic cells in the crypts, slight blunting of villi and loss of goblet cells. Infiltrating cells in the mesentery and bowel consisted mainly of CD 4+ cells, CD 8- T-cells and monocytes/macrophages. Lactulose-mannitol urinary excretion ratio was significantly increased in short-term cyclosporin treated allografts at days 50 and 100 posttransplant. Serum albumin levels were significantly lowered in this group at both time points examined. We developed two models in which CR occurs after small bowel transplantation. Long-term cyclosporin treatment delayed the development of CR, since functional abnormalities were only seen in the animals that were treated with short-term cyclosporin.

Increased expression of basic fibroblast growth factor during chronic rejection in intestinal transplants is associated with macrophage infiltrates.

Long-term survival of intestinal transplants is hampered by chronic rejection (CR). Since transplants with CR demonstrate fibrotic changes, the cytokine basic fibroblast growth factor (bFGF) may be involved in the tissue remodelling of chronic intestinal rejection. The aim of this study was to investigate the bFGF gene and protein expression and distribution in chronically rejecting intestinal allografts. Orthotopic small bowel transplantation was performed in the allogeneic DA-to-AS rat combination. Cyclosporin was administered temporarily to prevent acute rejection. Controls were DA isografts and normal DA. bFGF gene expression was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) of the ileum RNA and was standardized against Glyceraldehyde-3-phosphate-dehydrogenase (GAP-DH) expression. bFGF protein was determined using immunohistochemistry. To identify the bFGF-positive cell type, sequential sections were stained for cell markers. Allografts showed histological features of CR, whereas isografts preserved normal architecture. bFGF gene expression was present in normal ileum and significantly upregulated in allografts. Immunohistochemical staining showed a significant increase in bFGF protein compared to isografts. Most bFGF-positive cells were localized in the submucosa and muscularis, particularly around the neural plexus. bFGF-positive cells appeared to be ED-2-positive macrophages, strongly suggesting that the site of bFGF production is the activated macrophage. This study demonstrates increased bFGF mRNA and protein in chronically rejecting intestinal allografts that appear to be produced by macrophages.

Endothelial activation and cytokine expression in human acute cardiac allograft rejection.

By extrapolation from the responses of cultured human umbilical vein endothelial cells (EC) and bovine aortic EC to short-term cytokine stimulation, EC activation is postulated as a likely component of the host response in acute allograft rejection and cardiac transplant-associated accelerated arteriosclerosis. To investigate the extent to which EC activation occurs in vivo in humans and to identify potential targets for therapeutic interventions, we compared the phenotypic characteristics of vascular EC as seen during clinicopathologically significant vs non-significant acute cardiac allograft rejection. We used monoclonal and monospecific polyclonal antibodies to coagulation molecules [tissue factor, thrombomodulin (TM), antithrombin III (AT-III), fibrinogen/fibrin, cross-linked fibrin and von Willebrand factor (vWF)], adhesion molecules (P-selectin, ICAM-1) and major histocompatibility complex (MHC) class I and II molecules. In addition we sought evidence of local cytokine production (IL-1, IL-2R, IL-4, IL-6, IL-7, IL-8, TNF-alpha, PDGF-AA, PDGF-BB), which might mediate alterations in expression of these proteins. We found that in clinically significant grades of cardiac allograft rejection requiring increased immunosuppression, in contrast to lesser grades of rejection not requiring clinical intervention, there was increased microvascular EC activation and differential expression of cytokines. EC changes associated with more extensive cardiac allograft rejection requiring treatment included: (i) disruption of the normal anticoagulant state with downregulation of TM and AT-III, upregulation of tissue factor and vWF expression, and associated extensive fibrin deposition; (ii) upregulation of MHC class I antigens, which are potential targets for host cytotoxic T lymphocytes; (iii) increased expression of the leucocyte adhesion molecules P-selectin and ICAM-1; (iv) expression of the pro-inflammatory cytokines IL-1 beta and TNF-alpha; and (v) increased expression of PDGF-AA and BB, which are known to promote migration and proliferation of intimal cells, and hence may contribute to development of transplant-associated atherosclerosis. Collectively these findings suggest that immune events resulting in EC surface changes and/or production of key cytokines play a role in the pathogenesis of acute transplant rejection and may contribute to the long-term complication of accelerated arteriosclerosis in allograft coronary arteries.

Preservation of denervated muscle by sensory protection in rats.

The goal of this study was to determine whether sensory motor nerve crossover could alter post-denervation atrophy of skeletal muscle. Sixty adult Lewis rats were divided into three groups: 1) unilateral transection of the tibial nerve alone; 2) unilateral transection of the tibial nerve with immediate repair; and 3) unilateral tibial and sural nerve transections with repair of the proximal sural nerve (sensory) to the distal tibial nerve (motor). The unoperated hind legs acted as positive controls. At 1 and 2 months postoperatively, posterior compartment musculature was harvested, weighed, then fixed and stained for histologic analysis. One month postoperatively, mean muscle weight in Group 1 animals (transection alone) was 23.0 +/- 2.6 percent of the control side; for Group 2 animals (motor-motor repair) was 40.9 +/- 42 percent; and for the sensory-protected Group 3 animals (sensory-motor repair) was 26.7 +/- 2.8 percent of controls (n = 15 per group). Two months postoperatively, the mean weights were 14.5 +/- 0.9 percent, 58.8 +/- 7.3 percent, and 21.1 +/- 3.1 percent of controls for Groups 1, 2, and 3, respectively (n = 5 per group). Differences between groups were statistically significant. Histologic analysis of Group 1 specimens revealed generalized atrophy of all muscle fibers. In Group 2, specimens showed evidence of reinnervation and less atrophy. Group 3 specimens demonstrated an atrophic pattern with islands of non-atrophic fibers scattered throughout. Sensory protection was thus shown to have significant effect on post-denervation atrophy in rat skeletal muscle.

Comparison of 810 nm and 1064 nm wavelengths for interstitial laser photocoagulation in rabbit brain.

BACKGROUND AND OBJECTIVE: This laboratory animal study is a comparison of Nd:YAG 1064 nm and diode 810 nm laser wavelengths in brain interstitial laser photocoagulation (ILP). Specific goals were to identify potential complications and physical characteristics of the thermal damage at both wavelengths prior to undertaking a clinical trial in humans. STUDY DESIGN/MATERIALS AND METHODS: A total of 41 ILP illuminations were performed in vivo in the brains of 33 anesthetized rabbits using plane-cut fiber tips implanted directly or through catheters, and diffusing fiber tips. Delivered powers ranged from 1.1 to 4.2 W. Exposures ranged from 300 to 900 s. Survival ranged from 0 to 48 h. Experiments were performed in animals with and without VX-2 brain tumors. RESULTS: Thermal damage from 1.1 W at 810 nm was similar to that from 1.6 W at 1064 nm, but more pronounced. With plane-cut fiber tips, there was a greater propensity for severe physical effects (smoke, charring, bubbling, surface damage) at 810 nm than at 1064 nm, yet hemorrhage, thrombosis and vapor dissemination were observed at both wavelengths, in both normal brain and tumor. CONCLUSIONS: For ILP in brain, 1064 nm may be better suited than 810 nm, although both are questionable with plane-cut-fiber tips. Compactness and portability may be the only valid reasons for using laser diodes operating around 810 nm. At 1064 nm, the power delivered from plane-cut fiber tips should be less than 1.5 W, necessitating long exposures, or else an open catheter should be used. Fiber tips with distributed emission may be preferred, provided structural integrity can be maintained.

Altered expression of fibrogenic growth factors in IgA nephropathy and focal and segmental glomerulosclerosis.

The profile of fibrogenic growth factor expression was assessed in biopsies from 27 patients with IgA nephropathy (IgAN), 14 focal and segmental glomerulsclerosis (FSGS) patients and 8 controls, by immunohistochemistry. Increased platelet-derived growth factor (PDGF)-A and PDGF-B expression was detected in glomeruli and in vascular structures and collapsed tubules in the interstitium. Computer assisted image analysis demonstrated increased glomerular PDGF-A in IgAN (P < 0.05), but not FSGS patients, compared to controls, suggesting an association with mesangial proliferation. PDGF receptors were prominent in areas of mesangial expansion and intertubular fibrosis. Significant increases in interstitial PDGF Receptor beta (PDGFR-beta) were detected for both IgAN (P < 0.01) and FSGS (P < 0.05) patients. Interstitial PDGFR-beta expression was significantly correlated to monocyte/macrophage infiltrate (P < 0.0001). Increased basic fibroblast growth factor (bFGF) expression was observed segmentally in glomeruli, and in areas of tubulointerstitial damage. Higher proportions of patients with FSGS than IgAN had elevated interstitial bFGF (P < 0.005) and PDGF, reflecting the more severe degree of vascular and tubulointerstitial injury in FSGS patients. This study demonstrates distinct patterns of fibrinogenic growth factors in IgAN and FSGS, strongly associated with the severity and type of injury.

Localization of 11 beta-hydroxysteroid dehydrogenase type II in human epithelial tissues.

The enzyme 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2) confers specificity on the renal mineralocorticoid receptor by inactivating glucocorticoids. Mutations in this gene give rise to the syndrome of apparent mineralocorticoid excess, a congenital condition characterized by sodium retention, severe hypertension, and often by growth retardation. It is not known whether 11 beta HSD2 or another enzyme confers specificity in nonrenal sodium transporting epithelia, such as those in the sweat gland, salivary gland, and gastrointestinal tract. We previously have used the HUH23 antibody to localize 11 beta HSD2 in the human kidney, vascular smooth muscle cells, and placenta. In the present study, we have examined a range of human epithelia for the presence of 11 beta HSD2. In the skin, staining was seen in eccrine sweat glands and arterioles, whereas weak HUH23 immunostaining was observed in the epidermis. Staining was absent from sebaceous glands and hair follicles. In the parotid gland, the 11 beta HSD2 enzyme was present in striated and excretory ducts, whereas in the submandibular gland, it was found in striated and interlobular ducts. Acini, adipocytes, and associated tumor tissue did not stain with the HUH23 antibody. In the gastrointestinal tract, HUH23 stained ileal enterocytes, colonic absorptive cells, and epithelial goblet cells, whereas the rectum contained areas of staining and nonstaining absorptive cells. Gastrointestinal structures, such as the lamina propria, Peyer's patch, and goblet cells within the crypts of Lieberkuhn did not stain with the antibody. This study demonstrates the presence of 11 beta HSD2 in nonrenal sodium-transporting epithelia and describes a range of tissues affected in the syndrome of apparent mineralocorticoid excess.

Familial progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a degenerative neurological disease not typically associated with a family history. Two siblings developed identical clinical features consisting of supranuclear vertical ophthalmoplegia, bradykinesia, rigidity, gait disturbance, and dementia. There was no history of encephalitis or of exposure to known chemicals. L-dopa and dopamine agonist therapy were minimally effective. Autopsy of 1 patient revealed the typical pathological findings of PSP: severe neuronal loss with neurofibrillary tangles (NFTs) in the substantia nigra, subthalamic nucleus, and locus ceruleus. Prominent neurofibrillary degeneration of the amygdaloid nucleus and hippocampus was also observed. Scattered neurofibrillary tangles were seen in the cerebral cortices. Cerebellar degeneration was characterized by a loss of neurons in the dentate nucleus associated with neurofibrillary tangles. Lewy bodies and cortical neuritic plaques were notably absent. The existence of a rare familial form of PSP is supported by these 2 siblings.

Vascular localization of the 11 beta-hydroxysteroid dehydrogenase type II enzyme.

1. The enzyme 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2) confers specificity on the non-specific mineralocorticoid receptor by converting cortisol to cortisone. 2. We have examined the localization of this enzyme in the human skin, myocardium and saphenous vein by immunohistochemical techniques. 3. High amounts of 11 beta HSD2 immunoreactivity were found in smooth muscle cells in the arterioles of the skin, heart and saphenous vein. Lower amounts of staining were also found in longitudinal and concentric smooth muscle cells lining the lumen of the saphenous vein.

The sensitivity of normal brain and intracranially implanted VX2 tumour to interstitial photodynamic therapy.

The applicability and limitations of a photodynamic threshold model, used to describe quantitatively the in vivo response of tissues to photodynamic therapy, are currently being investigated in a variety of normal and malignant tumour tissues. The model states that tissue necrosis occurs when the number of photons absorbed by the photosensitiser per unit tissue volume exceeds a threshold. New Zealand White rabbits were sensitised with porphyrin-based photosensitisers. Normal brain or intracranially implanted VX2 tumours were illuminated via an optical fibre placed into the tissue at craniotomy. The light fluence distribution in the tissue was measured by multiple interstitial optical fibre detectors. The tissue concentration of the photosensitiser was determined post mortem by absorption spectroscopy. The derived photodynamic threshold values for normal brain are significantly lower than for VX2 tumour for all photosensitisers examined. Neuronal damage is evident beyond the zone of frank necrosis. For Photofrin the threshold decreases with time delay between photosensitiser administration and light treatment. No significant difference in threshold is found between Photofrin and haematoporphyrin derivative. The threshold in normal brain (grey matter) is lowest for sensitisation by 5 delta-aminolaevulinic acid. The results confirm the very high sensitivity of normal brain to porphyrin photodynamic therapy and show the importance of in situ light fluence monitoring during photodynamic irradiation.

Immunohistochemical localization of the 11 beta-hydroxysteroid dehydrogenase type II enzyme in human kidney and placenta.

It has been proposed that the inactivation of glucocorticoids by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) is an obligatory step in the kidney, permitting binding of aldosterone to the mineralocorticoid receptor, and in the placenta, protecting the fetus from high circulating levels of maternal glucocorticoids. Both low and high affinity isoforms of 11 beta HSD are known to exist, with evidence accumulating that the former species (11 beta HSD1) does not fulfill criteria that would allow it to perform these physiological functions. We have recently cloned a high affinity isoform of the enzyme (11 beta HSD2) from a human kidney library and have shown this species to possess all of the characteristics predicted from whole cell studies. In the present study we have raised a polyclonal antibody (HUH23) to a synthetic peptide deduced from the carboxy-terminus of the protein. The immunopurified antibody recognized a single band at 41,000 daltons on Western blots of mammalian cells transfected with an expression plasmid containing 11 beta HSD2, slightly smaller than the predicted 44,140 daltons protein. A single band of identical size was also seen in blots of human kidney and placenta, suggesting post-translational processing of the enzyme. Immunohistochemical studies on frozen sections of human kidney showed strong 11 beta HSD2 immunoreactivity in the cortical distal convoluted tubules and collecting ducts. Strong staining was also observed in medullary tubules, which had the appearance of collecting ducts and the thick ascending limb of Henle's loop. Staining of medium intensity was observed in vascular smooth muscle cells. Epithelial cells of glomeruli showed weak but detectable reactivity with HUH23. In the placenta, HUH23 antibody immunoreactivity was restricted to syncytial trophoblast cells in which strong staining was observed. These results suggest that the 11 beta HSD2 enzyme colocalizes with the mineralocorticoid receptor in the distal nephron where it allows aldosterone to occupy its physiological receptor. Furthermore, 11 beta HSD2 is also ideally situated in the placenta to protect the fetus from high circulating levels of maternal glucocorticoids.

Analysis of proliferating cell nuclear antigen expression aids histological diagnosis and is predictive of progression of human cardiac allograft rejection.

A quantitative immunohistological analysis was undertaken of 558 sequential paraffin-embedded and 22 snap-frozen endomyocardial biopsies (EMBs) from nine consecutive patients undergoing cardiac transplantation and followed for up to 1 year post-surgery. Serial monitoring was performed to assess whether 1) the phenotypic characteristics, 2) level of immune activation, or 3) expression of proliferation-associated antigens by intragraft leukocytes were useful in determining the grade of rejection or predicting further rejection episodes. Particular attention was given to those patients whose most recent EMBs showed grade 2 rejection, because these patients present a common problem in clinical management wherein a decision must be made as to whether or not to increase immunosuppression. Comparison of EMBs displaying various grades of rejection showed that whereas the absolute number of leukocytes (CD45), memory T cells (UCHL1/CD45RO), helper T cells (OPD4), and macrophages (Mac387) increased with increasing grade of rejection, the proportions of each subset remained similar. Cell proliferation was determined by labeling with monoclonal antibodies to proliferating cell nuclear antigen (cyclin) and Ki-67, and immune activation was assessed using an anti-interleukin-2 receptor (CD25) monoclonal antibody. The numbers of intragraft proliferating cell nuclear antigen-positive Ki-67+ or interleukin-2 receptor-positive cells were found to increase with increasing grades of rejection. Moreover, comparison of EMBs with equivalent histological features of rejection (grade 2) showed significantly (P < 0.0001) greater numbers of proliferating cell nuclear antigen-positive cells in EMBs preceding an episode of higher grade or persisting rejection versus EMB from patients whose rejection resolved, as seen on subsequent biopsy, without increased immunosuppression. These data suggest that the identification of proliferating or immunologically activated cells may aid in the histological diagnosis of clinical rejection and provide a valuable indicator predictive of likely further rejection episodes of increasing severity if grade 2 rejection is left untreated.

Mechanism of a clinically relevant protocol to induce tolerance of cardiac allografts. Perioperative donor spleen cells plus cyclosporine suppress IL-2 and interferon-gamma production.

Many groups have reported that preoperative injection of donor-derived whole spleen cells or major histocompatibility complex antigens prolongs organ allograft survival in experimental models, but the immunosuppressive mechanism(s) responsible remains unclear. A central, confounding issue is how to reconcile documentation of comparable levels of mRNA for IL-2 in suppressed versus control groups with obvious host hyporesponsiveness. We used a model of tolerance induction involving perioperative injection of donor spleen cells and injection of CsA at day 2 after transplant to analyze the serial expression of several proinflammatory cytokines relevant to development of alloresponsiveness within cardiac allografts and recipients' spleens. Four experimental groups of Lewis rats receiving vascularized heterotopic cardiac allografts from Brown Norway (BN) donors were evaluated: (1) untreated controls; (2) animals receiving intraoperative injection of donor BN spleen cells; (3) those receiving a single injection of CsA on day 2 post-Tx; and (4) animals given the combination of intraoperative BN spleen cells and CsA on day 2 post-Tx. Graft survival was significantly prolonged in Lewis rats receiving the combined spleen cell/CsA therapy (mean 64 days, with 40% of grafts surviving > 100 days, n = 15) compared with acute rejection at about 8 days (range 6-13, n = 20) in each of the 3 control groups (P < 0.0001). By comparison with acutely rejecting allografts in the control untreated group at day 7 post-Tx, allografts in rats receiving the combined perioperative spleen cell/CsA treatment showed (1) significantly reduced graft cellularity and interstitial edema; (2) significantly decreased features of immune activation, including infiltration by mononuclear cells expressing IL-2R or proliferating cell nuclear antigen; (3) decreased intragraft expression of the cytokines IL-2 and IFN-gamma; and (4) suppression of endothelial activation as evidenced by both failure of up-regulation of intracellular adhesion molecule-1 and maintenance of thrombomodulin expression by graft endothelium. Analysis of sections of recipients' spleens showed that spleen cell/CsA therapy led to significant reductions versus untreated controls, in expression of IL-2, IFN-gamma, and IL-2R. Similarly, mixed lymphocyte response cultures showed that responder cells from rats receiving combined therapy proliferated by 93-95% less than untreated animals. Our results suggest that the efficacy of this clinically relevant protocol is associated with suppression of IL-2 or IFN-gamma protein production, and that in the absence of such molecules, it appears that T cell-receptor occupancy by alloantigens readily induces a state of anergy in vivo.

Reduction of dietary phosphorus absorption by phosphorus binders. A theoretical, in vitro, and in vivo study.

Antacids used to decrease phosphorus absorption in patients with renal failure may be toxic. To find more efficient or less toxic binders, a three-part study was conducted. First, theoretical calculations showed that phosphorus binding occurs in the following order of avidity: Al3+ greater than H+ greater than Ca2+ greater than Mg2+. In the presence of acid (as in the stomach), aluminum can therefore bind phosphorus better than calcium or magnesium. Second, in vitro studies showed that the time required to reach equilibrium varied from 10 min to 3 wk among different compounds, depending upon solubility in acid and neutral solutions. Third, the relative order of effectiveness of binders in vivo was accurately predicted from theoretical and in vitro results; specifically, calcium acetate and aluminum carbonate gel were superior to calcium carbonate or calcium citrate in inhibiting dietary phosphorus absorption in normal subjects. We concluded that: (a) inhibition of phosphorus absorption by binders involves a complex interplay between chemical reactions and ion transport processes in the stomach and small intestine; (b) theoretical and in vitro studies can identify potentially better in vivo phosphorus binders; and (c) calcium acetate, not previously used for medical purposes, is approximately as efficient as aluminum carbonate gel and more efficient as a phosphorus binder than other currently used calcium salts.